The potential use of modified hemoglobin as a substitute for red blood cells in transfusions is widely documented to fill a critical need in medical therapeutics. Circulating red blood cells serve to deliver oxygen to tissues. A decrease in red blood cells as the result of loss of blood, causes serious and irreversible damage to organs. Large losses are life-threatening. Red blood cells present problems with respect to administration (typing), storage, and infection (AIDS, hepatitis). Thus, a product that would replace red blood cells in transfusions is widely sought.
Hemoglobin is the oxygen-carrying component of the red cell. Unmodified hemoglobin is a tetrameric assembly of protein components consisting of two sets of paired subunits, each with a heme prosthetic group to which oxygen binds. Outside the cell its properties are such that it cannot be used as a replacement for the cellular material. The unmodified material dissociates into dimeric units outside the cell. The dissociated form is not a useful substance and causes problems in the kidney. Thus, a cross-link is necessary to hold the subunits together. Another problem is that hemoglobin will deliver oxygen to cells if it is first oxygenated and if its affinity for oxygen is lower than that of the target cell.
As naturally occurring materials in humans and animals, hemoglobins are not expected to be treated as a foreign substance by the immune system. Thus, if the properties of hemoglobin can be adjusted by chemical modification to introduce properties necessary for hemoglobin to be used as a red cell substitute, an important product will result.
In the red blood cell, a high endogenous 2,3-diphosphoglycerate (DPG) binds to hemoglobin and induces hemoglobin to exist in a state that has a sufficiently low affinity for oxygen to transfer oxygen to the tissues. In order to keep the low affinity state of hemoglobin, it is desirable to permanently introduce an effect similar to that of DPG since the DPG concentration in the circulation is very low. The cooperative binding of oxygen (as indicated by Hill coefficient) should be maintained in order to have efficient transfer occur. The chemical modifications should also not be reversible. Further, modifications should be readily and specifically performed so that the product can be well-characterized and conveniently prepared.
Methyl acetyl phosphate originally synthesized as a site-specific reagent for hydroxybutyrate dehydrogenase (Kluger and Tsui (1980) J. Org. Chem. 45, 2723) has been reported to have an affinity for the binding site for 2,3-diphosphoglycerate in hemoglobin (Ueno et al., Archives Of Biochemistry And Biophysics, Vol. 244, No. 2, 795 (1986) and Ueno et al., The Journal of Biological Chemistry, Vol. 264, No.21, 12344 (1989)). It has been documented that three residues in or near this cleft between the beta-chains are acetylated by this reagent. The acetylation of Val-1, Lys-82 and Lys-144 and the absence of the acetylation of any of the amino groups of the alpha-chain indicate the specificity of methyl acetyl phosphate in its reaction with hemoglobin (Ueno et al., Archives Of Biochemistry And Biophysics, Vol. 244, No. 2, 795 (1986). It has further been documented that methyl acetyl phosphate and other monoesters of acyl phosphates may be used as acetylating agents for nucleophilic groups on proteins (Kluger, R. and Tsui, W. C., Cell Biol., Vol.64, 434 (1986)).
Many cross-linking reagents have been used in an attempt to produce a modified hemoglobin with oxygen transport properties similar to whole blood. The bifunctional analog 2-nor-2-formyl-pyridoxal 5'-phosphate provides a means of cross-linking the hemoglobin tetramer between the beta-subunits and has been reported to reduce oxygen affinity (Benesch et al., Biochem. Biophys. Res. Comm. 63, 1123-1129 (1975), J. Mol. Biol. 115, 627-642 (1977), Proc. Natl. Acad. Sci. U.S.A. 81, 2941-2943 (1984)). However, the cross-linking reagent is difficult to synthesize.
Under deoxygenated conditions, bis(3,5-dibromosalicyl) fumarate reacts with hemoglobin selectively to cross-link the alpha subunits between Lys-.alpha..sub.1 99 and Lys-.alpha..sub.2 99. The oxygen transport characteristics of this product were found to be similar to whole blood (Snyder et al., Proc. Natl. Acad. Sci. U.S.A. 84, 7280-7284 (1987) and Chatterjee et al., J. Biol. Chem., Vol. 261, No.21, 9929-9937 (1986).
U.S. Pat. No. 4,584,130 to Bucci and Fronticelli-Bucci describes a stable cross-linked, stroma-free hemoglobin, having a physiologically acceptable oxygen affinity. The cross-linking reagents were produced from one of two starting compounds, 2,2'-sulfonyl-bis-acetonitrite or 2,2'-sulfonyl-bis-acetate, which were reacted with sodium borohydride in a nitrogen atmosphere. To the product of this reaction was added, for example methyl chloroformate or 1,1'-sulfonyl-bis-imidazole to produce methyl 2,2'sulfonyl-bis-cyanoacetate and 2,2'-sulfonyl-2,2'imidazole-N-sulfonyl-bis-acetonitrile respectively. These compounds cross-link hemoglobin by binding preferentially, but not exclusively, in the 2,3-diphosphoglycerate (DPG) binding site. Reactions outside the DPG-binding site produce intermolecular cross-links.
Walder U.S. Pat. No. 4,600,531 describes a cross-linked stroma-free hemoglobin product suitable for use as a blood substitute and plasma expander. Walder discloses the use of phenyl esters, preferably bis (3,5-dibromosalicyl) fumarate, to cross-link hemoglobin between the two alpha-99 lysyl residues. Deoxyhemoglobin is reacted with the cross-linker in the presence of an added polyanion, such as inositol hexaphosphate, to block competing reactions at other sites of the protein, such as the DPG-binding site. Walder also discloses the reaction of oxyhemoglobin with mono (3,5-dibromosalicyl) fumarate to introduce a negatively charged carboxylate within the DPG-binding site.